Total syntheses of (–)-macrocalyxoformins A and B and (–)-ludongnin C

The complex and diverse molecular architectures along with broad biological activities of ent-kauranoids natural products make them an excellent testing ground for the invention of synthetic methods and strategies. Recent efforts notwithstanding, synthetic access to the highly oxidized enmein-type ent-kauranoids still presents considerable challenges to synthetic chemists. Here, we report the enantioselective total syntheses of C-19 oxygenated enmein-type ent-kauranoids, including (–)-macrocalyxoformins A and B and (–)-ludongnin C, along with discussion and study of synthetic strategies. The enabling feature in our synthesis is a devised Ni-catalyzed decarboxylative cyclization/radical-polar crossover/C-acylation cascade that forges a THF ring concomitantly with the β-keto ester group. Mechanistic studies reveal that the C-acylation process in this cascade reaction is achieved through a carboxylation followed by an in situ esterification. Biological evaluation of these synthetic natural products reveals the indispensable role of the ketone on the D ring in their anti-tumor efficacy.

The complex and diverse molecular architectures along with broad biological activities of ent-kauranoids natural products make them an excellent testing ground for the invention of synthetic methods and strategies.Recent efforts notwithstanding, synthetic access to the highly oxidized enmein-type entkauranoids still presents considerable challenges to synthetic chemists.Here, we report the enantioselective total syntheses of C-19 oxygenated enmein-type ent-kauranoids, including (-)-macrocalyxoformins A and B and (-)-ludongnin C, along with discussion and study of synthetic strategies.The enabling feature in our synthesis is a devised Ni-catalyzed decarboxylative cyclization/radicalpolar crossover/C-acylation cascade that forges a THF ring concomitantly with the β-keto ester group.Mechanistic studies reveal that the C-acylation process in this cascade reaction is achieved through a carboxylation followed by an in situ esterification.Biological evaluation of these synthetic natural products reveals the indispensable role of the ketone on the D ring in their anti-tumor efficacy.
As part of our ongoing research program aimed at the collective total synthesis of bioactive and structurally diverse ent-kauranoids, leveraging meticulously designed radical cascade reactions 20,21 , we pursued a radical cascade approach to synthesize the distinct fused A/E1/E2 ring system found in the unexplored highly oxidized C19-oxygenated enmein-type ent-kauranoids, such as (-)-macrocalyxoformins A (5) and B (6), as well as (-)-ludongnin C (7).Despite the non-trivial challenges associated with constructing the twist-boat B lactone ring, we present herein our synthetic endeavors toward achieving their total synthesis.
Although the proposed reductive decarboxylative cyclization/ RRPCO/C-acylation cascade holds conceptual efficiency, it presents at least two considerable challenges: (i) a viable acylation reagent with proper reactivity has to be selected: if the acylation reagent is not reactive enough, then the carbanion 10 might be acylated by the starting material RAE 11; conversely, if the acylation reagent is too reactive, then could be reduced prior to the reduction of 11 or the radical precursor of 10 41 .(ii) the desired C-acylation has to override the O-acylation 42 .Approaching these challenges, we thought that CO 2 would be a good choice [43][44][45][46] ; however, considering the lability of the β-keto acid product, the additional esterification step, and the potential scalability issue in the early stage of the total synthesis, we prioritize the search for an appropriate acylation reagent.

Preparation of the precursors and optimization of the reductive decarboxylative cyclization/RRPCO/C-acylation cascade
The preparation of the RAE 11 commenced with the alcohol (R)-16 (Table 1), which could be easily prepared in 3 steps at decagram scale in 87% ee and 63% overall yield using Rawal's procedure 47 .Etherification of alcohol 16 with tert-butyl bromoacetate (NaOH, TBAB, quant.)provided tert-butyl ester 17.Removal of the carboxyl tert-butyl protection (TFA) provided carboxylic acid, which can be converted to an array of RAEs in good-to-excellent yields.
We began the investigation of the proposed reductive decarboxylative cyclization/RRPCO/C-acylation cascade using the canonical ester 11a as starting material (83% yield from tert-butyl ester 17),   NiBr 2 •DME as catalyst, and Zn as reductant.After extensive screening of potential acylation reagents (entries 1-6, Table 1), we were pleased to find that the desired product 9d could be produced in 27% yield when di-tert-butyl decarbonate (Boc 2 O) was employed, while other tested acylation reagents (12a-f) performed sluggishly.An extensive investigation of bipyridine ligands (entries 7 and 8), alternative catalysts (entries 9-11), and the reaction temperature (entries 12 and 13) were unfruitful.Eventually, we were happy to find that changing N-hydroxyphthalimide (NHPI) ester 11a to N-(acyloxy)−1,8-naphthalimide 11d increased the yield of 9d to 53% (49% isolated yield, entry 16).Additionally, it is noteworthy that this cascade reaction can be performed at a decagram scale, easily producing multigram quantities of 9d in one pot (42% isolated yield).

Mechanistic studies
Although our proposed reductive decarboxylative cyclization/RRPCO/ C-acylation cascade showed good efficiency in the synthesis of the bicyclic β-keto ester 9d, the use of Boc 2 O as the C-acylation reagent seems counterintuitive because it is typically used to introduce the Boc protecting group to amine functionalities, with only a few examples as a C-acylation reagent reported to date [48][49][50] .We, therefore, performed a series of experiments to gain further insight into the mechanism of this cascade reaction (Fig. 2).
The reductive radical cyclization/RRPCO sequence was inferred from our findings that (i) replacing Boc 2 O (6 equiv) with D 2 O (20 equiv) in the standard reaction conditions provided the only observed product 18 in 80% yield with >99% deuterium incorporation (Fig. 2a), indicating a sequence of decarboxylative 5-exo-trig radical cyclization and an RRPCO of C10 radical occurred; (ii) no 9d was detected when 18 was applied as the starting material instead of RAE 11d (Fig. 2b), excluding the possibility that 18 was the precursor of the acylation.Moreover, we found that Boc 2 O was not a good C-acylation reagent for 18, as treatment of 18 with a variety of bases (e.g.LDA, NaHMDS) followed by adding Boc 2 O only provided trace amounts of 9d and the C2 acylated isomer (Supplementary Fig. 6).
We next focused on identifying the source of the C10 ester group of 9d (Fig. 2c) via a series of isotope labeling experiments.Initially, 13 C labeled Boc 2 O was prepared [ 13 CO 2 (>99% 13 C), t BuOK, MsCl, pyridine, Supplementary Fig. 7] and used instead of Boc 2 O.We obtained 9d with 69% 13 C incorporation (35% yield, Fig. 2c, entry 1), which indicated that the C10 ester group was not fully derived from Boc 2 O. Consequently, we labeled the RAE group of 11d with 13 C (see Supplementary Figs.11 and 12 for its preparation) and subjected 13 C-11d to the standard reaction conditions, we detected the product 9d with 27% CO 2 released by the decarboxylation process.To further demonstrate this point, we performed the standard reaction under a 13 CO 2 (>99% 13 C) atmosphere (Fig. 2c, entry 3), and a 44% 13 C incorporation of product 9d was observed.
On the basis of these lines of evidence, a putative mechanism for the acylation process is proposed (Fig. 2e).The carbanion 10 (was generated through the reductive decarboxylative cyclization/RRPCO process) reacts with CO 2 produced by the decarboxylation, giving rise to the carboxylate 20.Esterification of 20 by Boc 2 O affords the desired β-keto esters 9d along with one t BuO -and two CO 2 51 .The released CO 2 could also be involved in the carboxylation of carbanion 10, accounting for the high efficiency of the acylation and the relatively low 13 C incorporation in the above isotope labeling experiments.Note that: (i) we detected >99% 13 C incorporation of 9d when Boc 2 O and RAE 11d were replaced by 13 C labeled Boc 2 O and 13 C-11d simultaneously in standard reaction conditions (Fig. 2c, entry 4), (ii) Additionally, we found that treatment of the carboxylic acid 19 with our standard conditions (Fig. 2d) could afford the esterification product 9d in 37% yield.Both experiments support the plausibility of our proposed reaction mechanism.
Armed with a comprehensive understanding of the mechanism and optimal conditions, we next briefly explored the generality of this cascade process (Fig. 2f).Intriguingly, displacing the methyl group on the existing C4 quaternary carbon to an ethyl group (9e), a benzyl group (9f), an allyl group (9g), or removal of the C4 methyl group (9h) did not compromise the yield.Furthermore, the substrates with substitutions at C3 and C2 were also amenable to this cascade reaction, delivering 9h-9j in synthetically useful yields.Notably, the incorporation of an aromatic ring at the α-position of the α, β-unsaturated ketones (C10) resulted in the formation of the O-acylated product 9k in high yield, rather than the expected C-acylated product, indicating a significant impact of steric hindrance on the C-acylation.
Building the E1 ring and the radicophile 13: challenges in decarboxylative Giese reaction With a reliable and scalable synthesis of 9d in hand, we set out to construct the E1 THF ring (Fig. 3a).Treatment of 9d with formalin in the presence of Yb(OTf) 3 gave rise to the C10 aldol product 21 in a completely stereoselective manner 52 .The high stereoselectivity is plausibly attributable to the influence of the axial methyl group at C4 20 .Subjection of 21 to the conditions reported by Suárez and co-workers (PIDA, I 2 , hν) forged the E1 THF ring smoothly 30 , producing 22 in good yield (61% from 21).Notably, a ring flip process occurred during the reaction.Reduction of the C1 ketone of 22 with a sterically hindered reducing reagent LiAl(O t Bu) 3 H afforded the desired alcohol 23 with excellent diastereoselectivity (9:1, 80%), while other less hindered reagents (e.g., NaBH 4 , DIBAL-H) proved to be unviable.Having secured access to 23, we rapidly prepared two precursors (24 and 25), which can be used to construct the C9-C10 bond via the decarboxylative Giese reaction: deprotection of the t Bu ester of 23 with trifluoroacetic acid (TFA), protection of the C1 alcohol and carboxylic acid with tertbutyldimethylsilyl (TBS) group and hydrolysis of TBS ester produced acid 24 (93%), which was sequentially activated with NHPI to afford the RAE 25 (66%).
In the subsequent assembly process utilizing decarboxylative Giese reaction, despite our diligent efforts, employing the radicophile enone 13 in conjunction with various photoredox decarboxylation conditions using acid 24 (see Supplementary Fig. 31 for details) 23,24 or different reductive decarboxylation conditions using RAE 25 (see Supplementary Fig. 32 for details) [25][26][27] only resulted in direct decarboxylation rather than the desired coupling product 29 (Fig. 3c).We initially ascribed this outcome to the significant steric hindrance arising from the γ-quaternary carbon of α, β-unsaturated 13.Notably, to our knowledge, no prior reports exist on the decarboxylative Giese reaction of tertiary radicals with α, β-unsaturated acceptors containing a γ-quaternary carbon.
In an effort to mitigate the steric hindrance associated with the radicophile, we chose to employ α, β-unsaturated lactone (S)-31 53 (Fig. 3d, see Supplementary Fig. 30 for its enantioselective preparation), which lacks substitution on the carbon adjacent to the reacting carbon centers of the radicophile.Notably, this approach could capitalize on the intrinsic configurations of C13 in (S)-31, potentially leading to the desired stereochemistry at C9. Subsequent B lactone ring construction could employ a lactonic Ireland-Claisen rearrangement 54 .However, none of the decarboxylative conditions utilizing acid 24 and RAE 25 yielded the desired coupling product with (S)-31, underscoring the pivotal role of steric effects caused by the substituents at the reacting carbon centers of the α, β-unsaturated ester.
We also investigated the intramolecular Giese reaction using acids 34-36 (Fig. 3e, see Supplementary Figs.34-36 for their preparations) and the RAEs thereof 37-39 (see Supplementary Fig. 38 for their preparations); unfortunately, we could not detect the desired coupling products.We speculated that the failures were due to (i) the C1-ester groups not favoring the cis configuration needed in the reaction transition state and (ii) the relatively high barrier of rotation around the ester C-O bond 55 to the desired reaction transition state.Furthermore, attempts to use the C10-acyl telluride as the tertiary radical precursor 56 also proved unsuccessful in both intermolecular and intramolecular Giese reactions (see Supplementary Figs.33 and 40 for details).

Completion of the total synthesis
Recognizing the pivotal role of the steric effects of the radicophile in constructing the C9-C10 bond, we chose to use the simplest acyclic unsaturated esters with no substitution on the reacting carbon centers 20,57,58 .As shown in Fig. 4, the protection of the C1-OH of 23 with a benzyl group (NaH, BnBr, 84%) followed by deprotection of the C10tert-butyl ester (TFA) and activation of the resulting acid with NHPI (DIC, DMAP) provided RAE 40 in an excellent yield (99%).The benzylprotected RAE 40 was not used in the investigate of the aforementioned intermolecular decarboxylative Giese reaction (Fig. 3), due to the presence of potential coupling products containing alkene group, which would be incompatible with the benzyl deprotection.Subjection of RAE 40 with 2,2,2-trifluoroethyl acrylate under Baran's decarboxylative Giese reaction conditions [Ni(ClO 4 ) 2 •6H 2 O, Zn, LiCl] successfully afforded the desired coupling product 41 (75% NMR yield) 26 .Subsequent removal of the benzyl group under hydrogenation conditions (H 2 , Pd/C), followed by a spontaneous lactonization, yielded lactone 42, whose structure was confirmed by X-ray crystallography.

Anticancer activity evaluation of the synthetic natural products
The α-methylenecyclopentanone system (D ring) in ent-kauranoids is recognized as a crucial pharmacophore for their antitumour activity 1 .Our synthesized natural products 5-7 exemplify this role effectively.We evaluated their impact on cell viability across nine cancer cell lines from five human tissues.Macrocalyxformin B (6) displayed significant broadspectrum anticancer activity at the micromolar level, as shown in Table 2. Conversely, macrocalyxformin A (5) and ludongnin C (7) exhibited negligible and weak activity, respectively, across the tested cancer cell lines, reaffirming the essential role of the α-methylenecyclopentanone moiety in anti-tumor activity.Notably, the comparatively stronger anti-tumor activity observed in compound 7, in contrast to compound 5, implies a potential non-covalent interaction facilitated by the ketone group, thus highlighting a promising direction for future exploration.

Discussion
In summary, we have achieved the enantioselective total syntheses of three C19-oxygenated enmein-type ent-kauranoids: (-)-macrocalyxoformins A (5) and B ( 6) and (-)-ludongnin C (7).The enabling basis for this total synthesis is a devised Ni-catalyzed reductive decarboxylative cyclization/RRPCO/C-acylation cascade that allowed efficient construction of the E2 THF ring and an adjacent βketo ester group, which served as a handle for the installation of the E1 THF ring and the B lactone ring.Our mechanistic investigations revealed that the acylation step in this cascade is realized by a carboxylation of carbanion followed by an in situ esterification.We anticipate that the reductive decarboxylative cyclization/RRPCO/ C-acylation cascade reaction we developed could be extended to the syntheses of other highly oxidized and polycyclic natural products.Evolutionary studies on radicophiles for decarboxylative  Giese reaction, aimed at constructing the C10 quaternary carbon, revealed the challenges posed by radicophiles with substituents at the reacting carbon centers.Further anti-tumor examination of these synthetic natural products highlighted the crucial role of the ketone on the D ring.

General procedure for the preparation of redox-active esters
To a cooled (0 °C) solution of the tert-butyl ester (1.0 equiv) in CH 2 Cl 2 (0.3 M) was added TFA (0.6 mL/mmol tert-butyl ester).The reaction mixture was warmed to room temperature and stirred for 3 h.The reaction mixture was concentrated directly, giving the carboxylic acid, which was used directly for the next step without further purification.Note: To rapidly remove TFA completely, the above crude product can be dissolved in toluene and concentrated under vacuum; this process can be repeated until no TFA can be detected by 19 F NMR.The N, N'diisopropylcarbodiimide (DIC, 1.2 equiv) was added dropwise to a cooled (0 °C) mixture of above carboxylic acid (1.0 equiv), AOH (NHPI or its analogs, 1.1 equiv), and 4-dimethylaminopyridine (DMAP, 0.3 equiv) in anhydrous CH 2 Cl 2 (0.2 M).After 4 h stirring at room temperature, the reaction mixture was directly concentrated under reduced pressure.Purification by flash column chromatography (silica gel) gave the RAE.
General procedure for the reductive decarboxylative cyclization/radical-polar crossover/C-acylation cascade In a glovebox, Boc 2 O (6.0 equiv) was added to the mixture of the RAE (1.0 equiv), NiBr 2 •DME (10 mol%), and Zn (3.0 equiv) in anhydrous N-mehtyl-2-pyrrolidone (NMP, 0.075 M) at room temperature.The reaction mixture was then moved out of the glove box and heated to 50 °C.After stirring for 14 h the reaction mixture was filtered through a pad of Celite®, and the filter was washed with EtOAc.The filtrate was washed with H 2 O (2 times), brine (1 time), whereby the aqueous layers were back-extracted with EtOAc (3 times).The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the crude product.Purification by column chromatography (silica gel) gave the product.

Cell culture
The human cell lines ME-180, U2OS, A549, HCT-116, SW756, HeLa, SiHa, HuH-7, and SK-CO-1 were obtained from Cell Resource Center, Peking Union Medical College (Beijing, China).All cell lines were confirmed to be mycoplasma-free by PCR.Regular adherent cell culture methods were used to culture cells in tissue-culture incubators with 5% CO 2 at 37 °C.A549 was grown in RPMI-1640 medium with 10% fetal bovine serum (FBS) and 2 mM L-glutamine.SK-CO-1 was grown in MEM medium with 10% FBS and 2 mM L-glutamine.All other cells were grown in DMEM medium with 10% FBS and 2 mM L-glutamine.

Cell viability assay
Three thousand cells in 100 μL of medium were plated per well in 96well flat clear bottom white polystyrene TC-treated microplates (Corning, USA).Then cells were dosed with a serial dilution of compounds with a D300e digital dispenser (Tecan, Männedorf, Switzerland).Cell survival was measured 72 h later using CellTiter-Glo luminescent cell viability assay kit (Promega, Madison, according to the manufacturer's instructions.Luminescence was recorded by EnVison multimode plate reader (PerkinElmer, Waltham, USA).IC 50 was determined with GraphPad Prism v8.0.2 using baseline correction (by normalizing to DMSO control), the asymmetric (four parameters) equation, and the least-squares fit.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Table 2 |
IC 50 values (μM) of synthetic natural products against 9 different cancer cell lines The numbers in bold in the table represent compound numbers.